Figure 3.

NF-κB/Notch–coactivated MZPs are transplantable and convert to myeloid cells in recipient mice. (A) Flow analysis of donor (CD45.2⁺) chimerisms in host (CD45.1⁺) mice (n = 5) 3 months after administration of TriB MZBs (upper left) or MZPs (upper right; 3 × 10⁶ per recipient in both cases). Unexpectedly, CD45.2⁺ MZPs lacked B220 and GFP expression (lower left) but were positive for myeloid markers Mac1 and Gr1 (lower right). (B) Time course of abundance of CD45.2⁺GFP⁺ donor BM cells in CD45.1 hosts 2 to 3 months after cell transfer. WBM cells (3 × 10⁶ per recipient) were transferred to lethally irradiated CD45.1 recipients, and GFP⁺ cells were enumerated by flow cytometry at monthly intervals. BM from normal mice (black) was used as control. Unlike donor B cells from NIK mice (green) or ICN mice (blue), B cells from TriB mice (red) exhibited self renewal, with a peak at 3 months after BM transplantation (n = 5 for all groups). (C) Representative FACS plots of peripheral blood from mice receiving TriB MZP transplants. The Mac1⁺Gr1⁺ phenotype of these cells is consistent with AML (n = 3). (D) Flow analysis showing that a significant fraction of converted myeloid leukemia cells shown in panel C express stemness marker cKit (n = 3). (E) PCR VDJ recombination assay demonstrating clonality of converted myeloid leukemia cells (Bswitch, lanes 4 and 5). In contrast, TriB whole splenocytes (lane 2) and flow-sorted CD45.2⁺ cells recovered from host mice that had received TriB MZPs on day 4.5 (lane 3) were polyclonal. Lane 1, size marker. Bottom, germ line fragment of a T-cell gene used as control. (F) Sequencing analysis of VDJ rearrangements from panel E, as described in Figure 2H (supplemental Figure 3B). (G) Immunoblots showing levels of CEBPα and PAX5 in TriB splenic B cells (TriB), normal splenic B cells (Con), converted myeloid leukemia cells (Bswitch), and TriB lymphoma cells (TriB[lym]). Actin was included as a loading control.